Sfemii, by STEMCELL - Product details

1

Purification and Culture of Primary AML Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Human tissue was obtained with the required ethical approval from the NHS National Research Ethics Committee and informed consent from patients. Patient bone marrow biopsies were obtained, and the AML cells purified using lymphoprep followed by CD34 MACS bead enrichment. Patient mutation details are in Table 1. Primary cells and PDX cells (patient 5 only) were cultured on human mesenchymal stem cells, in SFEMII (StemCell Technologies) supplemented with 1% Pencillin/Streptomycin, 1 µM UM729 (Stemcell Technologies), 750 nM SR1 (Stemcell Technologies), 150 ng/ml SCF, 100 ng/ml TPO, 10 ng/ml FLT3, 10 ng/ml IL3, 10 ng/ml GM-CSF (all cytokines from Peprotech). Where primary cells were frozen prior to use, they were allowed to recover for a week before performing phenotypic assays but sorted directly from defrost for gene expression analysis. Healthy CD34+ cells (Amsbio) were cultured in SFEMII with StemSpan CD34 Expansion Supplement (Stem Cell Technologies) and 500 nM UM729 for 1 week, then moved into the t(8;21) media for 24 h prior to setting up assays.

Kellaway S.G., Potluri S., Keane P., Blair H.J., Ames L., Worker A., Chin P.S., Ptasinska A., Derevyanko P.K., Adamo A., Coleman D.J., Khan N., Assi S.A., Krippner-Heidenreich A., Raghavan M., Cockerill P.N., Heidenreich O, & Bonifer C. (2024). Leukemic stem cells activate lineage inappropriate signalling pathways to promote their growth. Nature Communications, 15, 1359.

+ Open protocol

+ Expand

2

Monocyte Differentiation and Activation

Check if the same lab product or an alternative is used in the 5 most similar protocols

For MS1-induction experiments, bone marrow or peripheral mononuclear cells were cultured in SFEM II supplemented with 1X CC110 (StemCell Technologies) with or without the presence of 100 ng/mL LPS or Pam3CSK4 (Invivogen) for up to 4 days. For re-stimulation experiments, sorted monocytes were rested for 24 hours in RPMI-1640 supplemented with 10% heat-inactivated FBS and 1X Pen-Strep (Gibco), before adding 100 ng/mL LPS (Invivogen).

Reyes M., Filbin M.R., Bhattacharyya R.P., Billman K., Eisenhaure T., Hung D.T., Levy B.D., Baron R.M., Blainey P.C., Goldberg M.B, & Hacohen N. (2020). An immune cell signature of bacterial sepsis. Nature medicine, 26(3), 333-340.

+ Open protocol

+ Expand

3

Cytokine-driven AML and CB CD34+ culture

Check if the same lab product or an alternative is used in the 5 most similar protocols

Primary AML cells and CB CD34⁺ progenitors were cultured in SFEMII (StemCell Technologies, Canada) with 1% P/S, recombinant IL-3 (20 ng/ml), IL-6 (20 ng/ml), SCF (100 ng/ml), FLT3-L (100 ng/ml) and G-CSF (20 ng/ml) (PeproTech, NJ, USA). Leukemia cell lines were cultured as per standard protocols. DMSO (0.1%) was used as vehicle control. Cell growth after drug treatment was enumerated by PrestoBlue^TM Cell Viability Reagent (ThermoFisher Scientific, MA, USA).

Man C.H., Zeng X., Lam W., Ng T.C., Kwok T.H., Dang K.C., Leung T.W., Ng N.K., Lam S.S., Cher C.Y, & Leung A.Y. (2022). Regulation of proton partitioning in kinase-activating acute myeloid leukemia and its therapeutic implication. Leukemia, 36(8), 1990-2001.

+ Open protocol

+ Expand

4

Imetelstat Cytotoxicity in Pediatric AML PDX

Check if the same lab product or an alternative is used in the 5 most similar protocols

1 × 10⁵ mouse passaged pediatric AML PDX lines with >98% human CD45+ cells, or normal bone marrow samples were seeded in culture media (SFEMII obtained from StemCell Technologies, Cambridge, MA, USA, supplemented with 50 ng/mL SCF, 25 ng/mL FLT3, 10 ng/mL IL-3, 10 ng/mL IL-6 and 25 ng/mL TPO) per well in a 96-well plate. Imetelstat or mismatch control oligo were diluted in media at a concentration of 2× and added to the cells to make the final concentrations 1 µM, 2.5 µM, 5 µM, 10 µM, or 20 µM. The untreated cells were used as control. The cells were incubated for 96 h at 37 °C, 5% CO₂ before staining with BV785-human CD45, APC-human CD34, Pacific blue-human CD38, FITC conjugated annexin V and propidium iodide (PI). Stained cells were analyzed by flow cytometry to detect cells in the early stage of apoptosis (Annexin V+/PI−), later stage of cell death (Annexin V+/PI+) and surviving cells (Annexin V−/PI−) in human CD45+ cells and LSC population (CD34+/CD38low).

Barwe S.P., Huang F., Kolb E.A, & Gopalakrishnapillai A. (2022). Imetelstat Induces Leukemia Stem Cell Death in Pediatric Acute Myeloid Leukemia Patient-Derived Xenografts. Journal of Clinical Medicine, 11(7), 1923.

+ Open protocol

+ Expand

5

Lentiviral Transduction of HSCs and MPPs

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

LT-HSCs were resuspended in SFEMII (StemCell Technologies) supplemented with growth factors described previously (Holmfeldt et al., 2016 (link)): Stem cell factor (SCF; 10 ng/ml), thrombopoietin (TPO; 20 ng/ml), insulin-like growth factor 2 (IGF2; 20 ng/ml) and fibroblast growth factor (FGF; 10 ng/ml) (BioLegend or StemCell Technologies) along with 5ug/ml polybrene (Sigma) and 1000 MOI lentiviral supernatant. Cells were spun at 2500rpm for 60min then cultured at 37°C and 5% CO₂ for 48hrs. Transduced GFP⁺ cells were sorted on a FACSAria (BD Biosciences). MPP4 cells were transduced as above in media containing IMDM plus 10% FBS, interleukin-3 (IL-3, 10 ng/ml), interleukin-6 (IL-6, 10 ug/ml), interleukin-7 (IL-7, 20 ng/ul), SCF (100 ng/ml), leukemia inhibitory factor (LIF, 20 ng/ml) (Peprotech).

Khokhar E.S., Borikar S., Eudy E., Stearns T., Young K, & Trowbridge J.J. (2020). Aging-Associated Decrease in the Histone Acetyltransferase KAT6B is Linked to Altered Hematopoietic Stem Cell Differentiation. Experimental hematology, 82, 43-52.e4.

+ Open protocol

+ Expand

6

Expansion of NK and CAR-NK Cells from AML

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were grown in SFEM II (StemCell, Cambridge, MA) supplemented with the following cytokines; SCF, IL-6, TPO, FLT3, GM-CSF, G-CSF, IL-3 (20 ng/mL) and EPO (10 ng/mL). A de-identified primary AML sample was obtained from OSU Leukemia Tissue Bank consistent with Declaration of Helsinki. NK or CAR-NK cells at a target:effector ratio of 1:2 were cultured for either 6 or 24 h. Following culture, cells were prepared for staining as previously described (Behbehani et al., 2012 (link)).

Naeimi Kararoudi M., Likhite S., Elmas E., Yamamoto K., Schwartz M., Sorathia K., de Souza Fernandes Pereira M., Sezgin Y., Devine R.D., Lyberger J.M., Behbehani G.K., Chakravarti N., Moriarity B.S., Meyer K, & Lee D.A. (2022). Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV. Cell Reports Methods, 2(6), 100236.

+ Open protocol

+ Expand

7

Expansion of Human Hematopoietic Progenitors

Check if the same lab product or an alternative is used in the 5 most similar protocols

Human HPCs (Lin⁻CD34⁺ ) and HSCs (Lin⁻CD34⁺CD38⁻) were cultured in Stemspan serum-free medium II (SFEM II, StemCell Technologies), supplemented with low concentrations of growth factors (GFs) similar to those present in long-term BM culture stroma-conditioned medium [granulocyte-macrophage colony-stimulating factor (GM-CSF) 200pg/mL, leukemia inhibitory factor (LIF) 50pg/mL, granulocyte colony-stimulating factor (G-CSF) 1ng/mL, stem cell factor (SCF) 200pg/mL, macrophage-inflammatory protein-1α (MIP-1α) 200pg/mL, and interleukin-6 (IL-6) 1ng/mL]^{42 (link)}. Mouse BM LT-HSCs were cultured in SFEM II supplemented with 10ng/ml SCF and 10ng/ml TPO. Human Umbilical Vein Endothelial Cells (HUVEC) and K562 cells were recently purchased from Lonza and ATCC respectively. We confirmed that HUVEC cells are human CD31 positive by flow cytometry and that K562 cells are BCR-ABL positive by QPCR. These two cell lines were tested for mycoplasma contamination and both were negative. HUVEC cells were cultured in complete EGM-2 medium (Lonza) and mouse BM ECs were cultured in complete mouse endothelial cell medium (Cell biologics). K562 cells were cultured in RPMI-1640 medium supplemented with 10% FBS (Life Technologies). All cells were cultured at 37°C with 5% CO₂ and high humidity.

Zhang B., Nguyen L.X., Li L., Zhao D., Kumar B., Wu H., Lin A., Pellicano F., Hopcroft L., Su Y.L., Copland M., Holyoake T.L., Kuo C.J., Bhatia R., Snyder D.S., Ali H., Stein A.S., Brewer C., Wang H., McDonald T., Swiderski P., Troadec E., Chen C.C., Dorrance A., Pullarkat V., Yuan Y.C., Perrotti D., Carlesso N., Forman S.J., Kortylewski M., Kuo Y.H, & Marcucci G. (2018). Bone Marrow Niche Trafficking of miR-126 Controls Self-Renewal of Leukemia Stem Cells in Chronic Myelogenous Leukemia. Nature medicine, 24(4), 450-462.

+ Open protocol

+ Expand

8

Expansion of Human Hematopoietic Progenitors

Check if the same lab product or an alternative is used in the 5 most similar protocols

Human HPCs (Lin⁻CD34⁺ ) and HSCs (Lin⁻CD34⁺CD38⁻) were cultured in Stemspan serum-free medium II (SFEM II, StemCell Technologies), supplemented with low concentrations of growth factors (GFs) similar to those present in long-term BM culture stroma-conditioned medium [granulocyte-macrophage colony-stimulating factor (GM-CSF) 200pg/mL, leukemia inhibitory factor (LIF) 50pg/mL, granulocyte colony-stimulating factor (G-CSF) 1ng/mL, stem cell factor (SCF) 200pg/mL, macrophage-inflammatory protein-1α (MIP-1α) 200pg/mL, and interleukin-6 (IL-6) 1ng/mL]^{42 (link)}. Mouse BM LT-HSCs were cultured in SFEM II supplemented with 10ng/ml SCF and 10ng/ml TPO. Human Umbilical Vein Endothelial Cells (HUVEC) and K562 cells were recently purchased from Lonza and ATCC respectively. We confirmed that HUVEC cells are human CD31 positive by flow cytometry and that K562 cells are BCR-ABL positive by QPCR. These two cell lines were tested for mycoplasma contamination and both were negative. HUVEC cells were cultured in complete EGM-2 medium (Lonza) and mouse BM ECs were cultured in complete mouse endothelial cell medium (Cell biologics). K562 cells were cultured in RPMI-1640 medium supplemented with 10% FBS (Life Technologies). All cells were cultured at 37°C with 5% CO₂ and high humidity.

Zhang B., Nguyen L.X., Li L., Zhao D., Kumar B., Wu H., Lin A., Pellicano F., Hopcroft L., Su Y.L., Copland M., Holyoake T.L., Kuo C.J., Bhatia R., Snyder D.S., Ali H., Stein A.S., Brewer C., Wang H., McDonald T., Swiderski P., Troadec E., Chen C.C., Dorrance A., Pullarkat V., Yuan Y.C., Perrotti D., Carlesso N., Forman S.J., Kortylewski M., Kuo Y.H, & Marcucci G. (2018). Bone Marrow Niche Trafficking of miR-126 Controls Self-Renewal of Leukemia Stem Cells in Chronic Myelogenous Leukemia. Nature medicine, 24(4), 450-462.

+ Open protocol

+ Expand

9

Expansion of CD34+ HSPCs

Check if the same lab product or an alternative is used in the 5 most similar protocols

All human tissues used in this study received informed donor consent and their usage was tracked internally by Novartis Institutes for BioMedical Research (NIBR) Human Tissue Network. All experimental protocols involving human tissues were approved by NIBR Human Tissue Council, and all experiments involving human tissues were executed in full compliance to NIBR Human Tissue Council Research and Quality guidelines. Cryopreserved bone marrow derived CD34⁺ HSPCs were purchased from Lonza or AllCells and thawed according to vendors’ instructions. Cells were cultured at 37 °C 5% CO₂ in SFEM II supplemented with CC110 (StemCell Technologies), 0.75 μM StemRegenin 1 (StemCell Technologies), 50 nM UM171 (StemCell Technologies), 50 ng/mL human recombinant IL-6 (Peprotech), and Pen-Strep (Life Tech 15140-122). Cells were cultured for 3–5 days before being used in experiments.

Yen J., Fiorino M., Liu Y., Paula S., Clarkson S., Quinn L., Tschantz W.R., Klock H., Guo N., Russ C., Yu V.W., Mickanin C., Stevenson S.C., Lee C, & Yang Y. (2018). TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells. Scientific Reports, 8, 16304.

+ Open protocol

+ Expand

10

Expansion and Cryopreservation of HSPCs

Check if the same lab product or an alternative is used in the 5 most similar protocols

In order to pre-expand HSPCs, cells was seeded at LD (1 × 10⁵ cells/mL) in SFEM II (STEMCELL Technologies) or SCGM (CellGenix) to enable non-cGMP or cGMP conditions, respectively. Medium was supplemented with 20 µg/mL streptomycin and 20 U/mL penicillin (Gibco, Life Technologies), as well as 100 ng/mL of the growth factors Flt3-L, TPO, and SCF (PeproTech). In addition, medium was supplemented with the small molecule inhibitors StemRegenin 1 (1 µM, SR1, STEMCELL Technologies) and UM171 (35 nM, STEMCELL Technologies). Throughout the period of pre-expansion, cells were kept at LD (1–5 × 10⁵ cells/mL), and medium was replenished at least every third day. Cells were pre-expanded for up to 8 days before being cryopreserved using CryoStor10 (CS10, STEMCELL Technologies). Upon thawing, cells were validated for CD34 expression.

Laustsen A., van der Sluis R.M., Gris-Oliver A., Hernández S.S., Cemalovic E., Tang H.Q., Pedersen L.H., Uldbjerg N., Jakobsen M.R, & Bak R.O. (2021). Ascorbic acid supports ex vivo generation of plasmacytoid dendritic cells from circulating hematopoietic stem cells. eLife, 10, e65528.

+ Open protocol

+ Expand

Sfemii

Lab products found in correlation

Close spelling variants of this product

24 protocols using sfemii

Purification and Culture of Primary AML Cells

Monocyte Differentiation and Activation

Cytokine-driven AML and CB CD34+ culture

Imetelstat Cytotoxicity in Pediatric AML PDX

Lentiviral Transduction of HSCs and MPPs

Expansion of NK and CAR-NK Cells from AML

Expansion of Human Hematopoietic Progenitors

Expansion of Human Hematopoietic Progenitors

Expansion of CD34+ HSPCs

Expansion and Cryopreservation of HSPCs

About PubCompare

Ready to get started?